The invention relates to a cartenoid agent for inhibiting the conversion of epithelial cells to tumors and a method for such treatment. More particularly, the invention relates to a cartenoid agent for inhibiting the conversion of melanocytes to melanomas and a method foe such treatment.
Although the description which follows is specifically directed to melanocytes and melanomas, it will be clear to a person skilled in the art that similar comments apply with respect to other types of epithelial cells.
Melanomas originate from a change in normal skin cells, melanocytes, which produce the brown pigment melanin we recognise as tan. Moles freckles result from areas of the skin with many melanocytes.
Melanomas are caused generally by the exposure of skin to sunlight. Persons of fair complexion have the greatest risk especially those who develop moles.
The influence of light on melanocytes is one way by which they can be changed to grow and divide differently, possibly causing a melanoma. The melanomas may be malignant, spreading to other parts of the body. Melanomas which do not spread are called benign melanomas.
Although melanomas normally form on exposed skin they can start in places such as the mouth.
Melanomas grow in size and need to be surgically removed before they spread and invade other parts of the body. If the melanomas spread to the inner organs, removal and treatment is more difficult and chemotherapy or radiotherapy need to be employed. For this reason, if melanocytes can be inhibited from converting to melanomas, the associated problems with the treatment of melanomas can be similarly reduced.
It has been hypothesized that carotenoids and in particular beta-carotene may reduce the risk of breast, lung, colon, prostate and cervical cancer, heart disease and stroke and may retard macular degeneration. In this respect, one hypothesis is that in mammals beta-carotene is converted to vitamin A and vitamin A analogues or retinoids (see Moon RC: Comparative aspects of carotenoids and retinoids as chemopreventive agents for cancer. J Nutr 119:127-134, 1989). It is this pro-vitamin A activity and the ability to prevent oxidative damage that has made carotenoids and, in particular, beta-carotene a compound of interest in chemopreventive studies. For instance, anti-oxidants are used, amongst other things, to quench free radicals that are by-products of normal metabolism in cells.
Beta-carotene has also been used in the treatment of erythropoietic protoporphyria (EPP). EPP is a genetic disease causing an inadequacy in the metabolism of porphyrin compounds. It results in a rapid blistering of the skin on exposure to sunlight.
Although, the effectiveness of carotenoids has been hypothesized in reducing the risk of certain instances of cancer, no studies have demonstrated the possible inhibiting effect of carotenoids in the conversion of melanocytes to melanomas.
For this reason, investigations were undertaken to determine the efficacy of carotenoids in inhibiting or reducing the conversion of melanocytes to melanomas. As part of this investigation, it was necessary to consider the difficulties in the use of carotenoids in human applications because of the nature and chemical properties of carotenoids.
Carotenoids are lipophyllic and therefore not soluble in water in useful quantities. It is believed that they are transported in the bloodstream in conjunction with low density lipoproteins.
To date several in vitro studies have taken place to determine th effect of beta-carotene on normal and transformed cell types using solvents to solubilize the beta-carotene such as tetrahydrofuran, butanol, chloroform, hexane, dimethylsulfoxide, ethanol or in a liposome micelle. Previous liposome preparations have shown toxicity in cell line cultures as well as being limited in application (see Betram J S, Pung A, Churley M, et al: Divers carotenoids protect against chemically induced neoplastic transformation. Carcinogenesis 12:671-678, 1991; Hazuka M B, Prasad-Edwards J, Newman F, it al; Beta-carotene induces morphological differentiation and decreases adenylated cyclase activity in melanocyte cells in culture. J Am Coll Nutr 9:143-149, 1990; Schultz T D, Chew B P, Seatnan W R, et al: Inhibitory effect of conjugated dienoic derivatives of linolcic acid and beta-carotene on the in vitro growth of human cancer cells. Canc Letters 63:125-133, 1992; Schwartz J L, Shaklar G: The selective cytotoxic effect of carotenoids and a-tocopherol on human cancer cell lines In vitro. J Oral Maxillofac Surg 50:367-373, 1992; Schwartz J L, Tanaka J, Khandekar V, et al: Beta-Carotene and/or Vitamin E as modulators of alkylating agents in SCC-25 human aquamous carcinoma cells. Canc Chemother Pharmacol 29:207-213, 1992; Zhang L-X, Cooney R V, Bertram J S; Carotenoids enhance gap juctional communication and inhibit lipid peroxidation in C3H/10T1/2 cells; relationship to their cancer chemopreventive action. Carcinogenesis 12:2109-2114, 1991; and Zang L-X, Cooney R V, Bertram J S ; Carotenoids Up-regulate connexin 43 gene expression independent of their provitamin A or antioxidant properties. Canc Res 52:5707-5712, 1992). These solvents have been found to have a toxic effect which is dose dependent. These solvents are also incompatible with human blood or lymph for the purposes of intravenous or injectable preparations.
Thus, in administering carotenoids, care must be taken to use appropriate carriers. Accordingly we carried out in vitro studies to determine the efficacy of carotenoids in inhibiting the conversion of epithelial cells to tumors and more particularly, the conversion of melanocytes to melanomas.
According to the present invention, a carotenoid agent is provided for inhibiting the conversion of epithelial cells to tumors, the agent including an effective amount of a water insoluble carotenoid component in a suitable non-toxic carrier medium.
The invention also provides a method of inhibiting the conversion of epithelial cells to tumors, including the step of applying to the cells, an effective amount of a water insoluble carotenoid component in a suitable non-toxic carrier medium.
The invention further provides a method of treating epithelial cells, so as to inhibit their conversion into tumors, including the step of applying to the cells, an effective amount of a water insoluble carotenoid component in a suitable non-toxic carrier medium.
The epithelial cell may be of mammalian origin. Preferably, the cell is a skin cell. It is particularly preferred that the skin cell is a melanocyte. In an even more preferred form of the invention, the epithelial cell is a human cell, and in a particularly preferred form of the invention, a human melanocyte.
Preferably, the water insoluble carotenoid component includes beta-carotene. In a yet further preferred form of the invention, the water insoluble carotenoid component includes from 2% to 50% by weight of beta-carotene. It is further preferred that the water insoluble carotenoid component includes from 20% to 40% by weight of bets-carotene. Even more preferably, the water insoluble carotenoid component includes 30% by weight of beta-carotene.
Preferably, the carrier medium includes a suspending agent selected from the group comprising fatty acids, triglycerides lipids, non-saponifiable lipid preparations, soluble hydrocarbons and combinations thereof.
Preferably, the triglycerides lipids are selected from the group comprising fats and/or oils derived from plant sources. The fats and/or oils may be derived from plant sources (in which case, it is particularly preferred that seed oils, such as cotton seed oil, sunflower oil or combinations thereof are used), or from animal sources (such as meat and fish). Seed oils are particularly preferred, and soya bean oil is an especially preferred oil for use in the carrier medium.
Preferably further, the water insoluble carotenoid component constitutes from 0.1% to 10% by weight of the agent. It is particularly preferred that the water insoluble carotenoid component constitutes from 1% to 5% by weight of the agent.
The non-toxic carrier medium preferably also includes an emulsifier. Preferably, the emulsifier is selected from the group including TWEEN(copyright) (polysorbate), glycerol fatty acid esters and acetylated esters of fatty acids. Glycerol mono-oleate is a particularly preferred emulsifier.
Preferably, the non-toxic carrier medium also includes a water soluble dispersing agent. It is particularly preferred that the water soluble dispersing agent is a sugar or a polyol. It is especially preferred that the water soluble dispersing agent is selected from the group including sorbitol and glycerol.
In all forms of the invention, preferably, the effective amount of the carotenoid agent contacting the melanocyte cells is from 0.1 to 10.0 micrograms/ml and more preferably, 0.3 to 3.0 micrograms/ml. These concentrations are achieved by dilution with suitable diluents. More preferably the diluting solution is selected from media suitable for the growth of the cells, aqueous buffers, normal intravenous preparations (including isotonic saline or 5% dextrose solution) and blood serum and combinations of the foregoing.
The term xe2x80x9cmixturexe2x80x9d as used herein is intended to include various physical forms including emulsions, solutions and crystal suspensions.
The following examples demonstrate the effectiveness of carotenoid compositions in the treatment of melanocytes and the relative non-toxicity of those compositions.